# Enhanced Proteomic Coverage in Tissue Microenvironment by Immune Cell Subtype Library-Assisted DIA-MS

## TL;DR

A new method using immune cell libraries improves proteomic analysis of tumor immune cells, revealing immune suppression in colorectal cancer models.

## Contribution

A novel immune cell subtype-specific spectral library-assisted DIA-MS strategy enables deep and sensitive proteomic profiling of microscale tissues.

## Key findings

- The immune cell-enriched library increased quantified proteins from 6978 to 7419 in microscale tissue samples.
- The approach identified 841 immune function-related proteins and low-abundance proteins like CD1D1 and CD244.
- CRC mouse models showed downregulation of adaptive immune signaling pathways in mesenteric lymph nodes.

## Abstract

Immune cells that infiltrate the tumor microenvironment (TME) play crucial roles in shaping cancer development and influencing clinical outcomes and therapeutic responses. However, obtaining a comprehensive proteomic snapshot of tumor-infiltrating immunity in clinical specimens is often hindered by small sample amounts and a low proportion of immune infiltrating cells in the TME. To enable in-depth and highly sensitive profiling of microscale tissues, we established an immune cell-enriched library-assisted strategy for data-independent acquisition mass spectrometry (DIA-MS). Firstly, six immune cell subtype-specific spectral libraries were established from sorted cluster of differentiation markers, CD8+, CD4+ T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, and macrophages in murine mesenteric lymph nodes (MLNs), covering 7815 protein groups with surface markers and immune cell-enriched proteins. The feasibility of microscale immune proteomic profiling was demonstrated on 1 μg tissue protein from the tumor of murine colorectal cancer (CRC) models using single-shot DIA; the immune cell-enriched library increased coverage to quantify 7419 proteins compared to directDIA analysis (6978 proteins). The enhancement enabled the mapping of 841 immune function-related proteins and exclusive identification of many low-abundance immune proteins, such as CD1D1, and CD244, demonstrating high sensitivity for immune landscape profiling. This approach was used to characterize the MLNs in CRC models, aiming to elucidate the mechanism underlying their involvement in cancer development within the TME. Even with a low percentage of immune cell infiltration (0.25–3%) in the tumor, our results illuminate downregulation in the adaptive immune signaling pathways (such as C-type lectin receptor signaling, and chemokine signaling), T cell receptor signaling, and Th1/Th2/Th17 cell differentiation, suggesting an immunosuppressive status in MLNs of CRC model. The DIA approach using the immune cell-enriched libraries showcased deep coverage and high sensitivity that can facilitate illumination of the immune proteomic landscape for microscale samples.

•A spectral library resource of 7815 proteins across six primary immune cell subtypes.•Library-assisted DIA offers deep coverage for immune proteomics in microscale tissue.•Library-assisted DIA enables sensitive mapping of immunological proteins in TME.•Application in CRC mouse models revealed immunosuppression in mesenteric lymph nodes.

A spectral library resource of 7815 proteins across six primary immune cell subtypes.

Library-assisted DIA offers deep coverage for immune proteomics in microscale tissue.

Library-assisted DIA enables sensitive mapping of immunological proteins in TME.

Application in CRC mouse models revealed immunosuppression in mesenteric lymph nodes.

For highly sensitive immune cell profiling in the tumor microenvironment, six immune cell subtype-specific spectral libraries covering 7815 proteins were established as a community resource. The library-assisted single-shot data-independent acquisition strategy offers deep coverage and identification of low-abundance immunobiologic process-related proteins in microscale tissue (1 μg), providing comprehensive coverage of surface markers and key signaling pathways. Application in colorectal cancer mouse models revealed an immunosuppressive status in mesenteric lymph nodes, confirming the feasibility of the strategy for future clinical sample applicability.